Molecular Diversity of Human Respiratory Syncytial Virus before and during the COVID-19 Pandemic in Two Neighboring Japanese Cities.

Human respiratory syncytial viruses (HRSVs) are divided into subgroups A and B, which are further divided based on the nucleotide sequence of the second hypervariable region (HVR) of the attachment glycoprotein (G) gene. Understanding the molecular diversity of HRSV before and during the coronavirus disease 2019 (COVID-19) pandemic can provide insights into the effects of the pandemic on HRSV dissemination and guide vaccine development. Here, we analyzed HRSVs isolated in Fukushima Prefecture from September 2017 to December 2021. Specimens from pediatric patients were collected at two medical institutions in neighboring cities. A phylogenetic tree based on the second HVR nucleotide sequences was constructed using the Bayesian Markov chain Monte Carlo method. HRSV-A (ON1 genotype) and HRSV-B (BA9 genotype) were detected in 183 and 108 specimens, respectively. There were differences in the number of HRSV strains within clusters prevalent at the same time between the two hospitals. The genetic characteristics of HRSVs in 2021 after the COVID-19 outbreak were similar to those in 2019. HRSVs within a cluster may circulate within a region for several years, causing an epidemic cycle. Our findings add to the existing knowledge of the molecular epidemiology of HRSV in Japan. IMPORTANCE Understanding the molecular diversity of human respiratory syncytial viruses during pandemics caused by different viruses can provide insights that can guide public health decisions and vaccine development.

Spectral and theoretical analysis of derivatives of 1,2,3,3-tetramethyl-3H-indolium iodide (TMI), a highly selective derivatization reagent of cyanide, and their utility for the analysis of cyanide concentrations in beverages.

We develop a specific derivatization gas chromatography-mass spectrometry (GC-MS) method for cyanide using 1,2,3,3-tetramethyl-3H-indium iodide as the derivatization reagent. The derivative compounds were synthesized and characterized using 1H nuclear magnetic resonance (NMR), 13C NMR, and Fourier transform infrared (FT-IR) spectroscopy. The high selectivity of this derivatization for cyanide is supported by calculations and activation energy comparisons. We applied this method to pure water, green tea, orange juice, coffee cafe au lait, and milk. Derivatization was performed by diluting 20 µL of sample solution with 0.1 M NaOH and adding 100 µL of saturated borax solution and 100 µL of 8 mM TMI solution, each drink was completed in 5 min at room temperature, and selected ion (m/z = 200) monitoring analysis was linear (R2 > 0.998) at 0.15 to 15 µM, with detection limits of 4-11 µM were shown. This method is expected to be widely used in forensic toxicology analysis and can be applied to beverages, which are forensically important field samples.

Molecular epidemiology of human metapneumovirus before and after COVID-19 in East Japan in 2017-2022.

Human metapneumovirus (hMPV) is genetically classified into two major subgroups, A and B, based on attachment glycoprotein (G) gene sequences, and the A2 subgroup is further separated into three subdivisions A2a, A2b (A2b1), and A2c (A2b2). The appearance of subgroup A2c viruses carrying a 180- or 111-nucleotide duplication in the G gene (A2c180nt-dup or A2c111nt-dup) have been reported in Japan and Spain. The pandemic of coronavirus disease 2019 (COVID-19) disrupted the epidemiological kinetics of other respiratory viruses, including hMPV. In this study, we analysed the sequences of hMPV isolates obtained from 2017 to 2022 in Tokyo and Fukushima, i.e., before and after COVID-19. Subgroup A hMPVs were detected in 2017 to 2019, and most cases were A2c111nt-dup, suggesting there was continuous momentum of this clade, identical to the global situation. Subgroup B, but not subgroup A, viruses were detected in 2022, after the COVID-19 peak. Phylogenetic analysis showed that these resumed subgroup B viruses were closely related to the viruses detected in 2013 to 2016 in Yokohama and in 2019 in Fukushima, suggesting a reappearance of local endemic viruses in East Japan.

Nearly Complete Genome Sequences of 12 Types of Human Rhinoviruses Isolated from Pediatric Inpatients in Fukushima, Japan.

We reported nearly complete genomic sequences of 12 serotypes of human rhinoviruses (HRVs) isolated from pediatric inpatients in Fukushima, Japan using an air-liquid interface culture of human bronchial tracheal epithelial cells. We found that various serotypes of HRV circulated locally and simultaneously from 2018 to 2021.

Ten Nearly Complete Genome Sequences of Human Orthorubulavirus 4 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 10 nearly complete genomic sequences of human orthorubulavirus 4, also called human parainfluenza virus 4 (HPIV4), isolated from pediatric inpatients with respiratory infections in Fukushima, Japan, by using an air-liquid interface culture of human bronchial and tracheal epithelial cells.

Changes in virus detection in hospitalized children before and after the severe acute respiratory syndrome coronavirus 2 pandemic.

The impact of strengthening preventive measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the prevalence of respiratory viruses in children was examined. After the SARS-CoV-2 pandemic, the rate of multiple virus detection among hospitalized children decreased. Immediately after the SARS-CoV-2 pandemic, respiratory syncytial and parainfluenza viruses were rarely detected and subsequently reemerged. Human metapneumovirus and influenza virus were not consistently detected. Non-enveloped viruses (bocavirus, rhinovirus, and adenovirus) were detected to some extent even after the pandemic. Epidemic-suppressed infectious diseases may reemerge as susceptibility accumulates in the population and should continue to be monitored.

Epidemiological and clinical characteristics of infections with seasonal human coronavirus and respiratory syncytial virus in hospitalized children immediately before the coronavirus disease 2019 pandemic.

INTRODUCTION: Seasonal human coronavirus (HCoV)-229E, -NL63, -OC43, and -HKU1 are seasonal coronaviruses that cause colds in humans. However, the clinical characteristics of pediatric inpatients infected with HCoVs are unclear. This study aimed to compare and clarify the epidemiological and clinical features of HCoVs and respiratory syncytial virus (RSV), which commonly causes severe respiratory infections in children. METHODS: Nasopharyngeal swabs were collected from all pediatric inpatients with respiratory symptoms at two secondary medical institutions in Fukushima, Japan. Eighteen respiratory viruses, including RSV and four HCoVs, were detected via reverse transcription-polymerase chain reaction. RESULTS: Of the 1757 specimens tested, viruses were detected in 1272 specimens (72.4%), with 789 single (44.9%) and 483 multiple virus detections (27.5%). RSV was detected in 639 patients (36.4%) with no difference in clinical characteristics between RSV-A and RSV-B. HCoV was detected in 84 patients (4.7%): OC43, NL63, HKU1, and 229E in 25 (1.4%), 26 (1.5%), 23 (1.3%), and 16 patients (0.9%), respectively. Patients with HCoV monoinfection (n = 35) had a significantly shorter period from onset to hospitalization (median [interquartile range] days, 2 [1-4.5] vs. 4 [2-5]), significantly shorter hospitalization stays (4 [3-5] vs. 5 [4-6]), and more cases of upper respiratory infections (37.1% vs. 3.9%) and croup (17.1% vs. 0.3%) but less cases of lower respiratory infection (54.3% vs. 94.8%) than patients with RSV monoinfection (n = 362). CONCLUSION: Seasonal HCoV-infected patients account for approximately 5% of children hospitalized for respiratory tract infections and have fewer lower respiratory infections and shorter hospital stays than RSV-infected patients.

Development of a cost-effective laser diode-induced fluorescence detection instrument for cyanide detection.

Cyanide is highly toxic to humans and the environment. It is very important to develop an on-site system for the quantitative analysis of cyanide with high sensitivity and reliability. In this study, we developed a cyanide detection system based on the reaction of vaporized cyanide on a glass-fiber filter soaked in a mixture of naphthalene-2,3-dicarboxaldehyde (NDA)-taurine-borate solution. Although the reaction product was stable for at least 3 days at room temperature, the reaction product on the strip was quickly quenched within a few minutes by direct irradiation with 405 nm light. To overcome this problem, we fabricated a simple device designed to detect the fluorescence intensity immediately after inserting a reaction strip into the device. The linearity of the calibration was obtained over a range of 1-100 µM of cyanide with good repeatability. The device is cost-effective (~ $300) and powered by batteries; therefore, it is suitable for the on-site determination of cyanide in crude samples.

Thirteen Nearly Complete Genome Sequences of Human Bocavirus 1 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 13 genomic sequences of human bocavirus 1 isolated from pediatric inpatients in Fukushima, Japan, using an air-liquid interface culture of human bronchial tracheal epithelial cells. This work suggests the endemic circulation of a human bocavirus variant with a unique amino acid signature in Fukushima.

In Situ Pinpoint Photopolymerization of Phos-Tag Polyacrylamide Gel in Poly(dimethylsiloxane)/Glass Microchip for Specific Entrapment, Derivatization, and Separation of Phosphorylated Compounds.

An improved method for the online preconcentration, derivatization, and separation of phosphorylated compounds was developed based on the affinity of a Phos-tag acrylamide gel formed at the intersection of a polydimethylsiloxane/glass multichannel microfluidic chip toward these compounds. The acrylamide solution comprised Phos-tag acrylamide, acrylamide, and N,N-methylene-bis-acrylamide, while 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] was used as a photocatalytic initiator. The Phos-tag acrylamide gel was formed around the channel crossing point via irradiation with a 365 nm LED laser. The phosphorylated peptides were specifically concentrated in the Phos-tag acrylamide gel by applying a voltage across the gel plug. After entrapment of the phosphorylated compounds in the Phos-tag acrylamide gel, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was introduced to the gel for online derivatization of the concentrated phosphorylated compounds. The online derivatized DTAF-labeled phosphorylated compounds were eluted by delivering a complex of phosphate ions and ethylenediamine tetraacetic acid as the separation buffer. This method enabled sensitive analysis of the phosphorylated peptides.

High-throughput N-glycan screening method for therapeutic antibodies using a microchip-based DNA analyzer: a promising methodology for monitoring monoclonal antibody N-glycosylation.

N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process. In this study, we focused on the HTP methodology for N-glycan analysis using a commercial microchip electrophoresis-based DNA analyzer and demonstrated the feasibility of the workflow consisting of sample preparation and electrophoretic separation. Even if there is a demand to analyze up to 96 samples, the present workflow can be completed in a day without expensive instruments and reagent kits for sample preparation, and it will be a promising methodology for cost-effective and facile HTP N-glycosylation analysis while optimizing the manufacturing process and development for therapeutic antibodies.

Nylon Monofilament Mold Three-dimensional Microfluidic Chips for Size-exclusion Microchip Electrophoresis: Application to Specific Online Preconcentration of Proteins.

We present a lithography-free procedure for fabricating intrinsically three-dimensional microchannels within PDMS elastomers using nylon monofilament molds. We embedded nylon monofilaments in an uncured PDMS composite to fabricate straight channels of desired length, for use as molds to form the microchannels. Next, we fabricated two layer devices consisting of dialysis membranes, which preconcentrate specific proteins in accordance with molecular weight, in between two layers of PDMS substrates with embedded microchannels. Because of the membrane isolation, analyte exchange between two fluidic layers can be precisely controlled by an applied voltage. More importantly, given that only small molecules pass through the dialysis membrane, the integrated membrane is suitable for molecular sieving or size exclusion for a concentrator prior to microchip electrophoresis. Researchers can use our microchip design for online purification and preconcentration of proteins in the presence of excess reagent immediately after fluorescent labeling. This method's technical advantage is that three-dimensional microstructures, such as microchannels that have a circular cross-section, are readily attainable and can be fabricated in a straightforward manner without using specialized equipment. Our method is a low-cost, environmentally sustainable procedure for fabricating microfluidic devices, and will render microfluidic processes more accessible and easy to implement.

Incidence and Relapse Triggers of Childhood Idiopathic Nephrotic Syndrome between 2006 and 2016: A Population-Based Study in Fukushima, Japan.

Childhood idiopathic nephrotic syndrome (NS) is defined by proteinuria and hypoproteinemia. The incidence of childhood idiopathic NS varies with age, race, residential areas, and social conditions. In Japan, its incidence was estimated to be 6.49 cases/100,000 children. Our study aimed to investigate the incidence, characteristics, and rate of relapse of idiopathic NS in Fukushima between 2006 and 2016. Overall, 158 children aged from 6 months to 15 years old (65.8% male) developed idiopathic NS (median age at onset, 5.3 years). The peak age at onset was three years. The average annual incidence of childhood idiopathic NS was 5.16 (range, 3.47-9.26) cases/100,000 children. The highest incidence was in 2011, which was the year of the Great East Japan Earthquake and nuclear power plant accident, and reportedly caused psychological distress in the children at the time. Conversely, the five-year birth cohort showed minor difference from 2008 to 2012. The rate of incidence in males aged < 5 years was thrice greater than in females of the same age and almost the same for males and females aged 11-15 years. Of 507 total relapses in 115 NS children, common triggers of relapses were steroid discontinuation or reduction and infection. The average annual incidence of childhood NS based on the Fukushima population was lower than previously reported in Japan, and the annual incidence has changed over an 11-year period. These changes may be affected by social or environmental factors, including mental stress associated with lifestyle changes after the disaster.

A rapid and facile preparation of APTS-labeled N-glycans by combination of ion pair-assisted extraction and HILIC-SPE for routine glycan analysis.

Glycoanalytical technology is required for a wide variety of scientific research, including basic glycobiological pharmaceutical, and biomarker research. Although several innovative analytical techniques have been developed for these purposes, quantitative glycan analysis based on electrophoretic separation, has often been impeded by the lack of cost-effective and facile sample preparation approaches. Here, we developed a rapid and facile sample preparation workflow for cost-effective glycan analysis and demonstrated its use with fully automated microchip electrophoresis (ME). Purification of 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans was based on the combination of ion-pair assisted extraction (IPAE) with hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE). Compared to commonly used sample preparation methods, the IPAE/HILIC-SPE method undergoes minimal nonspecific loss and undesirable degradation of N-glycans during the purification step. Furthermore, our method required only 10 min, and the entire workflow, including glycan release, labeling, and concentration processes was completed within 4 h. Although the present system should be improved to enable analysis of more complex mixtures, ME-based separation of APTS-labeled N-glycans offers a fully automated operation including conditioning, sample loading, separation, and can be analyzed with a sample-to-sample throughput of 120 s in parallel processes. The present workflow is easy to implement, does not require expensive reagents and instruments and may be useful for glycoscientists across disciplines.

A new chemosensor for cyanide in blood based on the Pd complex of 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol.

A new indirect chemosensor for the detection of cyanide in blood is developed. 2-(5-Bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol, a yellow dye, forms a blue-coloured complex with palladium ions. The yellow colour of this complex is regained upon reaction with cyanide ions. The complex shows high selectivity for the detection of cyanide over 16 other anions. The system was applied to two different methods for the detection of cyanide in human whole blood. As a quantitative absorbance method, blood samples were mixed with acid, and the resulting vaporised hydrogen cyanide was absorbed in an alkaline solution containing the complex in a Conway cell. The resulting absorbance response of the solution at 450 nm is linear over the range 4-40 µM (R2 = 1.000), and the limit of detection is 0.6 µM. Furthermore, the complex-soaked paper is applicable as a test strip for cyanide detection. When a test strip is used with 0.5 mL of blood, the limit of detection is 15 µM. The detection limits of these two methods are below the toxic blood cyanide concentration (19 µM). Therefore, both methods allow the quantification and screening of cyanide in blood samples. Furthermore, the test strip is low cost and enables on-site analysis.

Age-Related Changes in O-Acetylation of Sialic Acids Bound to N-Glycans of Male Rat Serum Glycoproteins and Influence of Dietary Intake on Their Changes.

O-Acetylation of sialic acids has been widely found in eukaryotic cells. Such modifications of sialic acids are tissue-specific and seem to be developmentally regulated. In this study, we performed comprehensive analysis of age-related changes in the serum N-glycans of male rats using capillary electrophoresis (CE) and investigated the changes in the O-acetylation of sialic acids bound to N-glycans with aging and different diets. The present method offered sufficient resolution to assess the degree of O-acetylation of the N-glycans and allowed for the determination of the age-related changes in O-acetylation of sialic acids. Using the CE-based method, we found that the relative abundance of disialo-biantennary N-glycans modified with 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac) significantly increased with aging. In addition, the relative abundances of N-glycans with two Neu5,9Ac reversed to those of N-glycans with only Neu5Ac during 12 weeks. Next, we evaluated the influence of high-fat diet and food restriction on age-related changes in O-acetylation. Although the total amount of disialo-biantennary N-glycans increased with aging, age-related O-acetylation of sialic acids was suppressed by a high-fat diet. On the other hand, food restriction enhanced the O-acetylation of sialic acids, and the relative abundance of N-glycans with two Neu5,9Ac residues at 15 weeks of age was higher than that observed in the standard diet group. These findings suggest that the O-acetylation of sialic acids is closely related to changes in energy metabolisms such as glycolysis or fatty acid metabolism.

Analysis of 2-aminopyridine labeled glycans by dual-mode online solid phase extraction for hydrophilic interaction and reversed-phase liquid chromatography.

Quantitative analysis of glycans released from glycoproteins using high-performance liquid chromatography (HPLC) requires fluorescent tag labeling to enhance sensitivity and selectivity. However, the methods required to remove large amounts of excess labeling reagents from the reaction mixture are time-consuming. Furthermore, these methods, including solvent extraction and solid phase extraction (SPE), often impair quantitative analysis. Here, we developed an online sample cleanup procedure for HPLC analysis of 2-aminopyridine (AP)-labeled glycans using a six-port/two-way valve and two small columns: one packed with a strong cation exchange resin (SCX) and the other comprising ODS silica gel. AP-labeled glycans delivered from an injection port were separated from excess AP by passing through an SCX column (4.6 mm i.d., 1 cm long) regulated to 40°C. The AP-labeled glycans were trapped on an ODS column (4.6 mm i.d., 1 cm long) to further separate them from inorganic contaminants. By changing the valve position after 2 min to connect the ODS column to an analysis column, AP-labeled glycans trapped in the ODS column were eluted with an acetonitrile-containing eluent followed by hydrophilic interaction liquid chromatography (HILIC) separation on an amide column or reversed-phase mode separation on a C30 column. This method was successfully used to analyze N-linked glycans released from several glycoprotein samples.

A practical method for preparing fluorescent-labeled glycans with a 9-fluorenylmethyl derivative to simplify a fluorimetric HPLC-based analysis.

Analysis of glycans in glycoproteins is often performed by liquid chromatography (LC) separation coupled with fluorescence detection and/or mass spectrometric detection. Enzymatically or chemically released glycans from glycoproteins are usually labeled by reductive amination with a fluorophore reagent. Although labeling techniques based on reductive amination have been well-established as sample preparation methods for fluorometric HPLC-based glycan analysis, they often include time-consuming and tedious purification steps. Here, we reported an alternative fluorescent labeling method based on the synthesis of hydrazone and its reduction using 9-fluorenylmethyl carbazate (Fmoc-hydrazine) as a fluorophore reagent. Using isomaltopentaose and N-glycans from human IgG, we optimized the Fmoc-labeling conditions and purification procedure of Fmoc-labeled N-glycans and applied the optimized method for the analysis of N-glycans released from four glycoproteins (bovine RNase B, human fibrinogen, human α1-acid glycoprotein, and bovine fetuin). The complete workflow for preparation of fluorescent-labeled N-glycans takes a total of 3.5 h and is simple to implement. The method presented here lowers the overall cost of a fluorescently labeled N-glycan and will be practically useful for the screening of disease-related glycans or routine analysis at an early stage of development of biopharmaceuticals.

Effect of the Lactococcus Lactis 11/19-B1 Strain on Atopic Dermatitis in a Clinical Test and Mouse Model.

Some lactic acid bacteria (LAB) are known to improve atopic dermatitis (AD) through the regulation and stimulation of the host immune system. In this study, we found that ingestion of yogurt containing Lactococcus lactis 11/19-B1 strain (L. lactis 11/19-B1) daily for 8 weeks significantly improved the severity scoring of atopic dermatitis (SCORAD) system score from 38.8 ± 14.4 to 24.2 ± 12.0 in children suffering from AD. We tried to identify which LAB species among the five species contained in the test yogurt contributed to the improvement in AD pathology using an AD mouse model induced by repeated application of 1-fluoro-2, 4-dinitrobenzene (DNFB). AD-like skin lesions on the dorsal skin and ear were most improved by L. lactis 11/19-B1 intake among the five LAB species. In addition, analysis of CD4+ T cell subsets in Peyer's patches (PPs) and cervical lymph nodes (CLNs) indicated that the intake of L. lactis 11/19-B1 generally suppressed all subsets related to inflammation, i.e., Th1, Th2 and Th17, instead of activating the suppressive system, Treg, in the AD mouse model. Histological observations showed ingestion of L. lactis 11/19-B1 significantly suppressed severe inflammatory findings, such as inflammatory cell filtration, epidermal erosion and eosinophil infiltration. These results suggest that the immunomodulatory effects of L. lactis 11/19-B1 contribute to improvements in AD pathology.